Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones.

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure led to a series of novel compounds. These compounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in intact A431 cells was shown, with IC(50) values ranging form 0.3-1µM for compound 42, and 0.1-0.3µM for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel compounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib and erlotinib (Tarceva®).

Chimeric tyrosine kinase-HDAC inhibitors as antiproliferative agents.

Combined treatment with tyrosine kinase inhibitors (TKi) and additional drugs is emerging as a promising strategy for cancer therapy. TKi and histone-deacetylase inhibitors (HDI) are two classes of anti-tumor agents with distant mechanisms of action. We have designed and synthesized chimeric compounds, which comprise structural elements of the TKi imatinib, and of prototypical HDI compounds. These compounds retain TKi activity similar to imatinib, exemplified by the inhibition of the platelet-derived growth factor receptor, and c-Kit kinase in intact cells. In addition, the chimeric compounds have in vitro and cellular HDI activity, and potently inhibit growth of cancer cell lines, including that of imatinib-resistant cell lines. Chimeric molecules with combined TKi and HDI activity may simplify combination treatment and be applicable to overcome clinical resistance to TKi single-agent therapy.

Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer: specific role of class I histone deacetylases in vitro and in vivo.

PURPOSE: Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of tumors of the large intestine. However, despite the importance of these molecules, comparably little is known on expression patterns and functions of specific HDAC isoforms in colorectal cancer. EXPERIMENTAL DESIGN: We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model. RESULTS: We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA-based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did. CONCLUSIONS: The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.

Association of patterns of class I histone deacetylase expression with patient prognosis in gastric cancer: a retrospective analysis.

BACKGROUND: Although histone deacetylases (HDACs) are known to have an important regulatory role in cancer cells, and HDAC inhibitors (HDIs) have entered late-phase clinical trials for the treatment of several cancers, little is known about the expression patterns of HDAC isoforms in tumours. We aimed to clarify these expression patterns and identify potential diagnostic and prognostic uses of selected class I HDAC isoforms in gastric cancer. METHODS: Tissue samples from a training cohort and a validation cohort of patients with gastric cancer from two German institutions were used for analyses. Tissue microarrays were generated from tumour tissue collected from patients in the training group, whereas tissue slides were used in the validation group. The tissues were scored for expression of class I HDAC isoforms 1, 2, and 3. Overall expression patterns (gHDAC) were grouped as being negative (all three isoforms negative), partially positive (one or two isoforms positive), or completely positive (all isoforms positive), and correlated with clinicopathological parameters and patient survival. The main endpoints were amount of expression of each of the three HDAC isoforms, patterns of expression of gHDAC, effect of metastasis on expression of HDAC and gHDAC, and overall survival according to HDAC expression patterns. FINDINGS: 2617 tissue microarray spots from 143 patients in the training cohort and 606 tissue slides from 150 patients in the validation cohort were studied. 52 of the 143 (36%) gastric tumours in the training cohort and 32 of the 150 (21%) gastric tumours in the validation cohort showed nuclear expression of all three HDAC isoforms. 60 (42%) of tumours in the training cohort and 65 (43%) in the validation cohort expressed one or two isoforms in the nuclei, whereas 31 (22%) of tumours in the training cohort and 53 (35%) in the validation cohort were scored negative for all three proteins. gHDAC expression in both cohorts was higher when lymph-node metastases were present (p=0.0175 for the training group and p=0.0242 for the validation group). Survival data were available for 49 patients in the training group and 123 patients in the validation group. In the validation cohort, 3-year survival was 44% (95% CI 34-57) in the HDAC1-negative group, 50% (39-64) in the HDAC2-negative group, and 48% (34-67) in the gHDAC-negative group. 3-year survival decreased to 21% (11-37) when HDAC1 was positive, 16% (9-31) when HDAC2 was positive, and 5% (1-31) when gHDAC (all isoforms) were positive. Those patients highly expressing one or two isoforms (the gHDAC-intermediate group) had an estimated 3-year survival of 40% (29-56). In multivariate analyses, high gHDAC and HDAC2 expression were associated with shorter survival in the training cohort (gHDAC: hazard ratio [HR] 4.15 [1.23-13.99], p=0.0250; HDAC2: HR 3.58 [1.36-9.44], p=0.0100) and in the validation cohort (gHDAC: HR 2.18 [1.19-4.01], p=0.0433; HDAC2: HR 1.72 [1.08-2.73], p=0.0225), independent of standard clinical predictors. INTERPRETATION: High HDAC expression is significantly associated with nodal spread and is an independent prognostic marker for gastric cancer. Additionally, we postulate that immunohistochemical detection of HDAC as a companion diagnostic method might predict treatment response to HDIs, thereby enabling selection of patients for this specific targeted treatment in gastric cancer.

Class I histone deacetylases 1, 2 and 3 are highly expressed in renal cell cancer.

BACKGROUND: Enhanced activity of histone deacetylases (HDAC) is associated with more aggressive tumour behaviour and tumour progression in various solid tumours. The over-expression of these proteins and their known functions in malignant neoplasms has led to the development of HDAC inhibitors (HDI) as new anti-neoplastic drugs. However, little is known about HDAC expression in renal cell cancer. METHODS: We investigated the expression of HDAC 1, 2 and 3 in 106 renal cell carcinomas and corresponding normal renal tissue by immunohistochemistry on tissue micro arrays and correlated expression data with clinico-pathological parameters including patient survival. RESULTS: Almost 60% of renal cell carcinomas expressed the HDAC isoforms 1 and 2. In contrast, HDAC 3 was only detected in 13% of all renal tumours, with particular low expression rates in the clear cell subtype. HDAC 3 was significantly higher expressed in pT1/2 tumours in comparison to pT3/4 tumours. Expression of class I HDAC isoforms correlated with each other and with the proliferative activity of the tumours. We found no prognostic value of the expression of any of the HDAC isoforms in this tumour entity. CONCLUSION: Class I HDAC isoforms 1 and 2 are highly expressed in renal cell cancer, while HDAC 3 shows low, histology dependent expression rates. These unexpected differences in the expression patterns suggests alternative regulatory mechanisms of class I HDACs in renal cell cancer and should be taken into account when trials with isoform selective HDI are being planned. Whether HDAC expression in renal cancers is predictive of responsiveness for HDI will have to be tested in further studies.

Histone deacetylase inhibitors suppress the inducibility of nuclear factor-kappaB by tumor necrosis factor-alpha receptor-1 down-regulation.

Recently, the inhibition of histone deacetylase (HDAC) enzymes has attracted attention in the oncologic community as a new therapeutic opportunity for hematologic and solid tumors including non-small cell lung cancer (NSCLC). In hematologic malignancies, such as diffuse large B-cell lymphoma, the HDAC inhibitor (HDI), suberoylanilide hydroxamic acid (SAHA), has recently entered phase II and III clinical trials. To further advance our understanding of their action on tumor cells, we investigated the possible effect of HDI treatment on the functionality of the nuclear factor-kappaB (NF-kappaB) pathway in NSCLC. We found that in the NSCLC cell lines, A549 and NCI-H460, the NF-kappaB pathway was strongly inducible, for example, by stimulation with tumor necrosis factor-alpha (TNF-alpha). Incubation of several NSCLC cell lines with HDIs resulted in greatly reduced gene expression of TNF-alpha receptor-1. HDI-treated A549 and NCI-H460 cells down-regulated TNF-alpha receptor-1 mRNA and protein levels as well as surface exposure, and consequently responded to TNF-alpha treatment with reduced IKK phosphorylation and activation, delayed IkappaB-alpha phosphorylation, and attenuated NF-kappaB nuclear translocation and DNA binding. Accordingly, stimulation of NF-kappaB target gene expression by TNF-alpha was strongly decreased. In addition, we observed that SAHA displayed antitumor efficacy in vivo against A549 xenografts grown on nude mice. HDIs, therefore, might beneficially contribute to tumor treatment, possibly by reducing the responsiveness of tumor cells to the TNF-alpha-mediated activation of the NF-kappaB pathway. These findings also hint at a possible use of HDIs in inflammatory diseases, which are associated with the overproduction of TNF-alpha, such as rheumatoid arthritis or Crohn's disease.

Cell type-- dependent effects of Polo-like kinase 1 inhibition compared with targeted polo box interference in cancer cell lines.

Multiple critical roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. Plk1 contains two domains amenable for targeted interference: a kinase domain responsible for the enzymatic function and a polo box domain necessary for substrate recognition and subcellular localization. Here, we compare two approaches for targeted interference with Plk1 function, either by a Plk1 small-molecule enzyme inhibitor or by inducible overexpression of the polo box in human cancer cell lines. Inducible expression of the Plk1 polo box resulted in growth inhibition of RKOp27 human colon adenocarcinoma cells without obvious signs of mitotic abnormalities. A Plk1 kinase inhibitor in the same cell line arrested cells in mitosis with subsequent onset of apoptosis. Similarly, PC-3 human prostate cancer cells were growth inhibited on expression of the polo box. Prolonged expression of the polo box in these cells resulted in the occurrence of binucleated or multinucleated cells. In contrast, U2OS human osteosarcoma cells responded to overexpression of the polo box with a massive mitotic accumulation coinciding with the onset of apoptosis. Comparison of spindle formation revealed very similar mitotic abnormalities in polo box-overexpressing U2OS cells compared with U2OS cells treated with the Plk1 kinase inhibitor. We conclude that interference with polo box function and inhibition of Plk1 kinase activity can exert very similar phenotypic effects in certain cell lines but highly contrasting effects in others. This may point to subtle differences in the molecular machinery of mitosis regulation in cancer cells.

2-aroylindoles and 2-aroylbenzofurans with N-hydroxyacrylamide substructures as a novel series of rationally designed histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors are considered to be drugs for targeted cancer therapy and second-generation HDIs are currently being tested in clinical trials. Here, we report on the synthesis and biological evaluation of a novel HDAC inhibitor scaffold with the hydroxamate Zn(2+) complexing headgroup, selected from the 2-aroylindol motif. Inhibition of nuclear extract HDAC and recombinant HDAC 1 as well as induction of histone H3K(9+14) hyperacetylation mediated by E-N-hydroxy-(2-aroylindole)acrylamides or E-N-hydroxy-(2-aroylbenzofuran)acrylamides were studied. Moreover, the cytotoxic activity, the effects on the cell cycle, and histone H3S(10) phosphorylation of selected compounds were determined. By use of a panel of 24 different human tumor cell lines, mean IC(50) values of the most potent analogues 6c and 7b were 0.75 and 0.65 microM, respectively. The novel compounds were shown to be no substrates of the P-glycoprotein drug transporter. Comparable to N(1)-hydroxy-N(8)-phenyloctanediamide "2 (SAHA)", cells in the S phase of the cell cycle are depleted, with partial arrest in G1 and G2/M and finally induction of massive apoptosis.

Molecular alterations after Polo-like kinase 1 mRNA suppression versus pharmacologic inhibition in cancer cells.

Multiple roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. We have employed chimeric antisense oligonucleotides to investigate the molecular alterations after targeted interference with Plk1 in RKO human colon adenocarcinoma and PC3 prostate cancer cells. Suppression of Plk1 mRNA resulted in a dramatic increase of the mitotic index followed by the onset of apoptosis. Mitotically arrested cells displayed randomly separated condensed chromosomes and the occurrence of multiple spindle poles with well-formed asters. Induction of apoptosis was strictly dependent on cell cycle progression: Genetically engineered RKO cells with inducible expression of the cyclin-dependent kinase inhibitor p27(Kip1) were completely refractory to Plk1 depletion-induced apoptosis when they were arrested in the G1 phase of the cell cycle. Various mitotic markers, including MPM-2, cdc25c, cyclin B1, or phosphorylated histone H3, were investigated to explore the molecular consequences of Plk1 depletion. Whereas most marker proteins showed similar alterations compared with treatment with paclitaxel, cdc25c was fully phosphorylated solely in paclitaxel-treated cells but only partially phosphorylated in Plk1-depleted cells, although both treatments caused a profound mitotic arrest. This differential phosphorylation of cdc25c was used to test whether a pharmacologic inhibitor of Plk1 would exert the same cellular effects as interference with Plk1 on a mRNA level. It was found that the differential electrophoretic mobility of cdc25c can serve as a reliable molecular marker to track inhibition of Plk1 by small-molecule inhibitors within a cell.

[4-(imidazol-1-yl)thiazol-2-yl]phenylamines. A novel class of highly potent colchicine site binding tubulin inhibitors: synthesis and cytotoxic activity on selected human cancer cell lines.

Synthesis and cytotoxic activity in the submicromolar range of a series of [4-(imidazol-1-yl)thiazol-2-yl]phenylamines are described. Cell cycle dependent cytotoxicity on RKO human colon carcinoma cells with inducible expression of p27(kip1) and the influence on microtubule formation were investigated. Considering the significant correlation between the IC(50) values of tubulin polymerization inhibition, [(3)H]colchicine competition, and cytotoxicity of the investigated compounds, tubulin is the main cellular target. The inhibition of microtubule formation was shown to be mediated by interference with the colchicine binding site of tubulin. In depth analysis of the investigated compounds allowed the identification of modifications that altered the pharmacological profile of the compounds from a mitosis-inducing phenotype to a G1 cell cycle arresting phenotype.

2-aroylindoles, a novel class of potent, orally active small molecule tubulin inhibitors.

2-Aroylindoles with 5-methoxy-1H-2-indolyl-phenylmethanone (D-64131) as the lead structure were discovered as a new class of synthetic, small molecule tubulin inhibitors. By competitively binding with [(3)H]colchicine to alphabeta-tubulin and inhibiting microtubule formation, cycling cells were arrested in the G(2)-M phase of the cell division cycle. The proliferation of tumor cells from 12 of 14 different organs and tissues was inhibited with mean IC(50)s of 62 nM and 24 nM by D-64131 and D-68144, respectively, comparable with the potency of paclitaxel with mean IC(50) of 10 nM. By measuring the cytotoxicity in a human colon carcinoma cell model with ectopic ecdysone-inducible expression of the cyclin-dependent kinase inhibitor p21(WAF1), specificity toward cycling cells was demonstrated. In contrast to microtubule inhibitors from natural sources, 2-aroylindoles did not alter the polymerization-dependent GTPase activity of beta-tubulin and are not substrates of the multidrug resistance/multidrug resistance protein efflux pump. No cross-resistance toward cell lines with multidrug resistance/multidrug resistance protein independent resistance phenotypes became evident. In animal studies, no signs of systemic toxicity were observed after p.o. dosages of up to 400 mg/kg of D-64131. In xenograft experiments with the human amelanoic melanoma MEXF 989, D-64131 was highly active with treatment resulting in a growth delay of 23.4 days at 400 mg/kg. Therefore, D-64131 and analogues have the potential to be developed for cancer therapy, replacing or supplementing standard therapy regimens with tubulin-targeting drugs from natural sources.

Switching off HER-2/neu in a tetracycline-controlled mouse tumor model leads to apoptosis and tumor-size-dependent remission.

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (> or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.

Synthesis and cytotoxic activity of 2-acyl-1H-indole-4,7-diones on human cancer cell lines.

Synthesis and cytotoxic activity of a series of 2-acyl-1H-indole-4,7-diones on human cancer cell lines are described. Due to close structural relationship to 2-acylindoles, potent inhibitors of tubulin polymerization, the mode of action of these novel compounds has been investigated. Cytotoxicity, the influence on tubulin polymerization, and cell cycle dependent cytotoxicity on colon carcinoma cells by investigation of RKO exo p27 versus RKO p27(kip1) cells are described. IC50 values of arrested versus proliferating cells differ only in a range of two to fourfold and therefore cellular targets, predominantly relevant for mitotic progression, are excluded. As shown by the significant difference in the IC90 values on different tumor cell lines, the investigated compounds seem to act selectively on mammary and renal cancer cells.

Bis(1H-2-indolyl)methanones as a novel class of inhibitors of the platelet-derived growth factor receptor kinase.

The novel lead bis(1H-2-indolyl)methanone inhibits autophosphorylation of platelet-derived growth factor (PDGF) receptor tyrosine kinase in intact cells. Various substituents in the 5- or 6-position of one indole ring increase or preserve potency, whereas most modifications of the ring structures and of the methanone group as well as substitution at both indoles result in weak or no activity. An ATP binding site model, derived by homology from the FGFR-1 tyrosine kinase crystal structure suggesting hydrogen bonds of one indole NH and the methanone oxygen with the backbone carbonyl and amide, respectively, of Cys684, explains why only one indole moiety is open for substitution and locates groups in the 5- or 6-position outside the pocket. The hitherto most active derivatives, 39, 53 and 67, inhibit both isoforms of the PDGF receptor kinase in intact cells, with IC(50) of 0.1-0.3 microM, and purified PDGFbeta-receptor in vitro, with IC(50) of 0.09, 0.1, or 0.02 microM, respectively. PDGF-stimulated DNA synthesis is inhibited by these derivatives with IC(50) values of 1-3 microM. Kinetic analysis of 53 showed an ATP-competitive mode of inhibition. The compounds are inactive or weakly active toward a number of other tyrosine kinases, including the FGF receptor 1, EGF receptor, and c-Src kinase, as well as toward serine-threonine kinases, including different PKC isoforms and GRK2, and appear therefore selective for PDGF receptor inhibition.

Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance.

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.

Novel chimeric histone deacetylase inhibitors: a series of lapatinib hybrides as potent inhibitors of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and histone deacetylase activity.

Reversible lysine-specific acetylation has been described as an important posttranslational modification, regulating chromatin structure and transcriptional activity in the case of core histone proteins. Histone deacetylases (HDAC) are considered as a promising target for anticancer drug development, with 2a as pan-HDAC inhibitor approved for cutanous T-cell lymphoma therapy and several other HDAC inhibitors currently in preclinical and clinical development. Protein kinases are a well-established target for cancer therapy with the EGFR/HER2 inhibitor 5 approved for treatment of advanced, HER2 positive breast cancer as a prominent example. In the present report, we present a novel strategy for cancer drug development by combination of EGFR/HER2 kinase and HDAC inhibitory activity in one molecule. By combining the structural features of 5 with an (E)-3-(aryl)-N-hydroxyacrylamide motif known from HDAC inhibitors like 1 or 3, we obtained selective inhibitors for both targets with potent cellular activity (target inhibition and cytotoxicity) of selected compounds 6a and 6c. By combining two distinct pharmacologically properties in one molecule, we postulate a broader activity spectrum and less likelihood of drug resistance in cancer patients.

Design of chimeric histone deacetylase- and tyrosine kinase-inhibitors: a series of imatinib hybrides as potent inhibitors of wild-type and mutant BCR-ABL, PDGF-Rbeta, and histone deacetylases.

Inhibitors of histone deacetylases are a new class of cancer therapeutics with possibly broad applicability. Combinations of HDAC inhibitors with the kinase inhibitor 1 (Imatinib) in recent studies showed additive and synergistic effects. Here we present a new concept by combining inhibition of protein kinases and HDACs, two independent pharmacological activities, in one synthetic small molecule. In general, the HDAC inhibition profile, the potencies, and the probable binding modes to HDAC1 and HDAC6 were similar as for 6 (SAHA). Inhibition of Abl kinase in biochemical assays was maintained for most compounds, but in general the kinase selectivity profile differed from that of 1 with nearly equipotent inhibition of the wild-type and the Imatinib resistant Abl T(315)I mutant. A potent cellular inhibition of PDGFR and cytotoxicity toward EOL-1 cells, a model for idiopathic hypereosinophilic syndrome (HES), are restored or enhanced for selected analogues (12b, 14b, and 18b). Cytotoxicity was evaluated by using a broad panel of tumor cell lines, with selected analogues displaying mean IC(50) values between 3.6 and 7.1 muM.

A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.

Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.

Trithiocarbonates as a novel class of HDAC inhibitors: SAR studies, isoenzyme selectivity, and pharmacological profiles.

Inhibitors of histone deacetylases (HDAC) are currently developed for the treatment of cancer. These include compounds with a sulfur containing head group like depsipeptide, alkylthiols, thiocarboxylates, and trithiocarbonates with a carbonyl group in the alpha-position. In the present investigation, we report on the synthesis and comprehensive SAR analysis of HDAC inhibitors bearing a tri- or dithiocarbonate motif. Such trithiocarbonates are readily accessible from either preformed or in situ prepared alpha-halogenated methylaryl ketones. A HDAC isotype selectivity and a substrate competitive mode-of-action is shown for defined analogues. Exploration of the head group showed the necessity of the dithio-alpha-carbonyl motif for potent HDAC inhibition. Highly potent, substrate competitive HDAC6 selective inhibitors were identified (12ac:IC 50 = 65 nM and K i = 110 nM). Trithiocarbonate analogues with an aminoquinoline-substituted pyridinyl-thienoacetyl cap demonstrate a cytotoxicity profile and potency comparable to that of suberoylanilide hydroxamic acid (SAHA) as an approved cancer drug.